TRIP13 predicts poor prognosis in clear cell renal cell carcinoma

Abstract

What is the leading molecular mechanism that causes broad resistance to systemic therapies remains a key question in renal cancer related research. We explored associations of TRIP13 expression with the clinical course using the tissue microarray (TMA). The TMA contained specimens from 87 patients diagnosed with clear cell renal cell carcinoma (ccRCC). We performed immunohistochemistry to investigate TRIP13 protein expression levels. The overall survival (OS) was analyzed using the Kaplan-Meier method and log-rank statistics. Univariate and multivariate analyses were conducted using Cox proportional hazard models. Median follow up for the TMA cohort was 7.0 years. Tissues from 28.74% of patients demonstrated high TRIP13 expression. Mean TRIP13 expression in TRIP13-rich tumors was significantly higher comparing to adjacent normal tissues (P < 0.05). TRIP13 expression did not significantly correlate with stage nor tumor grade (P > 0.05). Elevated expression of TRIP13 served as an independent unfavorable prognostic indicator of survival in ccRCC (P < 0.05). TRIP13 overexpression predicts poor prognosis in ccRCC. Together with the emerging reports, this observation raises a suspicion that TRIP13 is a substantial driver of resistance to systemic therapies against kidney cancer.

Introduction

Renal cell carcinoma (RCC) is among the 10 most common cancers in both men and women, and its incidence is on the rise. In 2020, 73,750 new cases and 14,830 deaths due to RCC will occur in the US and over 400,000 new cases will occur worldwide [1]. Up to 30-40% of RCC cases are present as metastatic disease, either initially or after curative treatment [2]. The most common subtype, clear cell renal cell carcinoma (ccRCC), arises from the proximal convoluted tubule cells and accounts for approximately 70% of all cases [3]. Despite significant improvements in the clinical management over the last decade, most patients with metastatic ccRCC succumb to cancer progression within 1.5 years [4].

The exceptional intratumoral heterogeneity of RCC represents a considerable challenge limiting the efficacy of established systemic therapies [5]. Such treatment often further exacerbates the heterogeneity and leads to outgrowth of tumor cell subclones with resistance properties, including the resistance to apoptosis [6,7]. The accumulating alterations found in both intrinsic and extrinsic apoptotic pathways aberrantly extend cells viability and eventually contribute to cancer progression [8]. Since apoptosis causes negligible damage to adjacent tissues [9], the apoptotic pathway-targeted therapies emerge as particularly promising strategy for RCC treatment.

TRIP13 is a protein encoded by TRIP13 gene. Recent evidence implicates TRIP13 in various cell cycle phases, including meiosis, G2/Prophase and during the mitotic spindle assembly checkpoint (SAC) activation. TRIP13 is required for the development of higher-order chromosome structures and contributes to synaptonemal complex formation. It also promotes early steps of the DNA double-strand breaks (DSBs) repair process. The latest reports together with in silico analysis, indicate its prominent role in driving tumorigenesis.

The Human Pathology Atlas is based on a systems-based analysis of the transcriptome of 17 main cancer types using data from 8,000 patients [10]. A national supercomputer center was used to analyze more than 2.5 petabytes of underlying publicly available data from the Cancer Genome Atlas (TCGA) to generate 900,000 survival plots describing the consequence of RNA and protein levels on clinical survival. All the data in the knowledge resource allows exploration of the human proteome. In this study, we explore the clinical association of TRIP13 with ccRCC histology and oncologic outcomes using the tissue microarray (TMA) ccRCC cohort, and validate these findings in TCGA.

Materials and methods

Tissue microarray

Tissue microarray (TMA) slide was obtained from a commercial supplier (US Biomax, Rockville, MD; TMA catalog number HKid-CRC180Sur-01). The TMA (HKid-CRC180Sur-01) contained specimens from 92 patients, tumor and matched normal adjacent tissue (1 core/case), followed up for 7 years. Cores derived from 3 patients were missing, therefore these patients were excluded from the analysis. Retrievable patient data included age, pathology diagnosis, TNM, grade, stage and overall survival. The quality of the TMA was additionally approved by our pathologist. The study follows the principles of the Declaration of Helsinki. The tissues were collected under the highest ethical standards and HIPPA approved protocols with the donor being informed completely and with their consent. Since the tissues were commercially purchased, the study has been exempted from requiring ethical approval.

Immunohistochemistry

The TMA slide was processed at the Department of Clinical Pathology. The primary rabbit polyclonal anti-TRIP13 (HPA005727) antibody (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) was applied to estimate the expression of TRIP13 protein. The protocol has been standardized using a series of positive and negative control reactions. The positive control reaction was performed on a tissue model selected according to reference sources (The Human Protein Atlas: http://www.proteinatlas.org) and the antibody data-sheet. TRIP13 positive control reaction was performed on pancreatic cancer tissue showing cytoplasmic and nuclear expression. All negative control reactions were performed on additionally analyzed tissue sections, by substituting the primary antibody with a solution of 1% BSA (bovine serum albumin) diluted in PBS (phosphate buffered saline). Immunohistochemical staining was performed using primary rabbit polyclonal anti-TRIP13 (1:200) antibody and visualization system EnVisionFlex+ Anti-Mouse/Rabbit HRP-Labeled Polymer (Dako, Agilent Technologies) on an Autostainer Link48 platform. Finally, tissue sections were dehydrated in ethanol of increasing concentration (from 80% to 98%), then cleared in a series of xylenes (from I to IV) and cover-slipped in a medium (Dako, Agilent Technologies, USA).

IHC analysis and scoring

Initially, two experienced pathologists blinded to the clinical data evaluated the immunostained slides using the light microscope ELIPSE E800 (Nikon Instruments Europe, Amsterdam, Netherlands) at 20× and 40× original objective magnification. IHC revealed cytoplasmic and nuclear TRIP13 expression.

The cytoplasmic staining intensity of cells and percentage of cells at each staining intensity level were determined for each fixed core in the TMA. Staining intensity was graded as 0 (negative), 1+ (weak), 2+ (modarate), and 3+ (strong). The H-score was assigned using the following formula: [1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+)], obtaining a value from 0 to 300.

The nuclear expression evaluation was scored on a two-point scale: 0 (negative IHC reaction result) and 1 (positive IHC reaction result).

Statistical analysis

All the statistical analyses were performed using Statistica version 10 (StatSoft) and Microsoft Excel 2019. The comparative studies were analyzed statistically using the nonparametric chi-square test. The p value < 0.05 was considered statistically significant.

Results

The location and expression of TRIP13 protein in ccRCC TMA cohort

IHC was performed on 87 pairs of ccRCC and corresponding normal tissues. Five cores of corresponding tissues were lost during IHC staining procedure. Table 1 summarizes the characteristics of the TMA cohort. The mean age of patients was 59 years (range: 29-83 years) and the median follow-up was 7.0 years.

Table 1.

Baseline characteristics of TMA (n = 87) patient cohort

Clinical information	n (%)
Age, yr
Mean	59.0
Range	29-83
Stage
I	58 (66.67)
II	17 (19.54)
III	3 (3.45)
IV	2 (2.30)
Unknown	0 (0.00)
T Stage
T1	62 (71.26)
T2	17 (19.54)
T3	4 (4.60)
Unknown	4 (4.60)
Lymph nodes
N1	1 (1.15)
N0/Nx	84 (96.55)
Unknown	2 (2.30)
Metastasis
Yes	2 (2.30)
No	85 (97.70)
Grade
G1	32 (36.78)
G1-G2	14 (16.09)
G2	27 (31.03)
G2-G3	4 (4.60)
G3	9 (10.34)
G3-G4	1 (1.15)
Median follow up time	7.0
Disease course
Alive	59 (67.82)
Dead	28 (32.18)

Cytoplasmic TRIP13 staining was observed in 77 (88.51%) of 87 ccRCC tissues and the median expression was 100 (interquartile range 0-215). Among adjacent controls, 70 (85.37%) of 82 cores were positive and the median expression was 115 (interquartile range 70-200). Cytoplasmic expressions of TRIP13 in ccRCC tissues were lower than those in adjacent controls (P < 0.05, Figure 1A). Next, we dichotomized the cytoplasmic expressions of TRIP13 to low expression and high expression. Using the tool Cutoff Finder, we set the best cutoff at 105 [11]. Mean TRIP13 expression in TRIP13-rich tumors was significantly higher comparing to adjacent normal tissues (P < 0.05, Figure 1B). Similarly, adjacent normal tissues were characterized by elevated TRIP13 expression when compared to TRIP13-depleted tumors (P < 0.05) (Figure 1C).

Figure 1.

A. Cytoplasmic expression of TRIP13 in Clear Cell Renal Cell Carcinoma (CCRCC) and adjacent normal tissue (Control). B. Cytoplasmic TRIP13 expression in TRIP13-rich CCRCC and control. C. Cytoplasmic TRIP13 expression in TRIP13-depleted CCRCC and control.

Nuclear TRIP13 staining was observed in 14 cancer tissues (16.28%) (Figure 2A) and only in 2 adjacent normal tissues (2.44%) (Figure 2B). This difference was statistically significant (P < 0.05).

Figure 2.

Prevalence of positive TRIP13 nuclear expression among (A) clear cell Renal Cell Carcinoma tissues, and (B) adjacent normal tissues (Control).

Clinical course and TRIP13 protein expression in ccRCC TMA cohort

TRIP13 protein expression did not significantly correlate with TNM stage nor tumor grade (both P > 0.05). Univariate analysis revealed that patients with high cytoplasmic TRIP13 protein expression had significantly shorter OS comparing to those with low expression (P < 0.05, HR = 2.88 [1.35-6.15]) (Figure 3). We found no significant association between the presence of TRIP13 nuclear expression and OS (Figure 4). In conclusion, TRIP13 overexpression predicts poor prognosis in ccRCC. Together with the emerging reports, this observation raises a suspicion that TRIP13 is a substantial driver of resistance to systemic therapies against kidney cancer.

Figure 3.

The survival curve of clear cell Renal Cell Carcinoma patients according to TRIP13 cytoplasmic expression.

Figure 4.

The survival curve of clear cell Renal Cell Carcinoma patients according to TRIP13 nuclear expression.

Discussion

TCGA ccRCC cohort analysis

We found that cytoplasmic TRIP13 protein overexpression significantly correlates with poor survival in ccRCC patients. To evaluate whether the expression of TRIP13 mRNA was also associated with the clinical course of the disease, we accessed TCGA database. All transcriptomics information were obtained employing the Human Pathology Atlas, the separate part of The Human Protein Atlas available from www.proteinatlas.org. TCGA cohort consisted of 528 patients diagnosed with ccRCC [12]. The available characteristics of study subjects are summarized in Table 2. The mean age of patients was 60.5 years (range: 26-90 years) and the median follow-up was 3.28 years. The TCGA RNA-seq data was mapped using the Ensembl gene id available from TCGA, and the FPKMs (number Fragments Per Kilobase of exon per Million reads) for TRIP13 were subsequently used for quantification of expression with a detection threshold of 1 FPKM. Based on the FPKM value of TRIP13, patients were classified into two expression groups. To choose the best FPKM cutoff for grouping the patients most significantly, all FPKM values from the 20th to 80th percentiles were used to group the patients, significant differences in the survival outcomes of the groups were examined and the value yielding the lowest log-rank P value (3.4e-11) was selected. 109 of 528 (20.64%) patients had higher expression than the established cutoff. The prognosis of each group of patients was examined by Kaplan-Meier survival estimators and the survival outcomes of the two groups were compared by log-rank tests. The five-year survival was reached by 70% of patients with low TRIP13 expression and 39% of those with high expression. Taken together, TRIP13 mRNA expression is prognostic and its high expression is unfavourable in RCC (P < 0.05), according to TCGA.

Table 2.

Baseline characteristics of TCGA (n = 528) patient cohort

Clinical information	n (%)
Age, yr
Mean	60.5
Range	26-90
Sex
Male	344 (65.15)
Female	184 (34.85)
Race
White	459 (86.93)
Black or African American	54 (10.23)
Asian	8 (1.52)
Unknown	7 (1.33)
Stage
I	263 (49.81)
II	57 (10.80)
III	123 (23.30)
IV	82 (15.53)
Unknown	3 (0.57)
Median follow up time	3.28
Disease course
Alive	355 (67.23)
Dead	173 (32.77)

The Human Protein Atlas tissue repository could not be used to evaluate TRIP13 protein expression in RCC because it showed low or negative immunoreactivity to both recommended antibodies (HPA053093 and HPA005727). Also the group of patients in this trial was small (n = 12).

Role of TRIP13 in cancer

TRIP13 takes part in a variety of cellular activities, including cell cycle regulation, DNA repair and apoptosis. Study on multiple myeloma cells revealed that overexpression of TRIP13 abrogates SAC. The underlying mechanism includes activation of PI3K-Akt signaling pathway that induces proteasome-mediated degradation of MAD2, the key component of SAC [13]. Dysfunctional SAC contributes to chromosomal instability (CIN), aneuploidy, and eventually facilitates cancer progression [14-16]. Moreover, one of major downstream effectors of AKT is the mammalian target of rapamycin (mTOR), which induces cell growth, proliferation, survival, and motility, as well as angiogenesis [17].

According to study of Yao et al., TRIP13 promotes growth and metastasis of hepatocellular carcinoma through inhibition of TGF-β1/Smad3 signaling [18]. Repressed Smad3 activity has been associated with breast cancer bone metastasis by its effects on tumor angiogenesis, and epithelial-mesenchymal transition (EMT) [19]. Zhou et al. revealed that TRIP13 enhances the proliferation and invasion via activation of the NOTCH signaling and induction of EMT [20]. In damaged cells, TRIP13 functions to favor non-homologous end joining (NHEJ) over homologous recombination (HR). Both are the major pathways for DNA DSBs repair. While HR results in accurate repair, NHEJ is an intrinsically error-prone pathway and may lead to CIN and eventually carcinogenesis [21].

Study of Banerjee et al. demonstrated significant impact of TRIP13 in chemoresistance development among head and neck cancers. Cells with downregulated TRIP13 expression, treated with cisplatin were characterized by better response rate and slower growth [22]. TRIP13 might enhance the resistance to systemic therapy in RCC as well. The systemic therapy (targeted therapy, immunotherapy or chemotherapy) could constitute a selective pressure acting on TRIP13 expression in RCC cells. During the course of the disease, the population of cells with higher expression of TRIP13 would rise because of its protective properties. Ultimately, the cancer tissue would become irreversibly resistant to applied treatment. Our results do not support this hypothesis, because we did not find significant relationships between protein expression and grade or stage of the disease. On the other hand, this approach might be worth pursuing, since the relatively small cohort of advanced ccRCC within our TMA could not ensure a valid representation.

Although the detrimental effect of TRIP13 has been confirmed in biologically diverse neoplasms [13-25], the exact mechanisms and their relative importance are not yet clear. While, as previously described, TRIP13 promotes malignant properties of ovarian cancer cells [20], elevated TRIP13 mRNA expression is associated with favorable outcomes in ovarian cancer patients [12]. It demonstrates that the actual contribution of TRIP13 to tumorigenesis could be far more complex.

Role of TRIP13 in kidney

It remains unexplored whether TRIP13 plays a significant role in renal cell carcinoma. However, Pressly et al. recently shed new light on the antiapoptotic role of TRIP13 in renal tubules. As they reported, TRIP13 interacts with Tetratricopeptide Repeat Domain 5 (TTC5) and inhibits p53. Insufficient TRIP13 consequently increases the susceptibility of damaged tubular epithelial cells to progress towards apoptotic cell death [31].

Interestingly, biallelic loss-of-function mutations in TRIP13 have been shown to predispose to Wilms tumor, a kidney cancer that primarily affects children [32]. The authors of this report indicate a substantial impairment of SAC, which eventually leads to a high rate of chromosome missegregation in these patients. This study supports the existence of a close relationship between TRIP13 and SAC, which when disturbed, increases cancer risk or drives its progression.

Potential role of TRIP13 in renal cell carcinoma

In Figure 5, we summarized probable interactions among TRIP13 and other molecules within kidney cancer cell. This pathway diagram illustrates how TRIP13 may affect survival in these patients, and therefore may serve as a starting point for translational research.

Figure 5.

Probable interactions among TRIP13 and other molecules within clear cell Renal Cell Carcinoma. Overexpression of TRIP13 activates PI3K/AKT/mTOR pathway. AKT induces proteasome-mediated degradation of MAD2, the key component of SAC. Dysfunctional SAC leads to CIN and aneuploidy, which, together with mTOR, mediate cancer progression. TRIP13 together with TTC5 as a cofactor, inhibits p53 signaling and, consequently, suppresses the apoptosis. In cells with damaged DNA, TRIP13 functions to favor NHEJ over HR. NHEJ as more likely to be inaccurate, may contribute to cancer progression. TRIP13 also induces the expressions of E-cadherin and vimentin directly or through activation of the NOTCH signaling. The net effect is the promotion EMT, which is directly associated with gain of migratory and invasive capabilities. TRIP13 reduces the expressions of TGF-β1, TβRII and Smad3, the mediators of cellular senescence. The inhibition of TGF-β1/Smad3 signaling supports tumor growth. Decreased Smad3 activity promotes EMT. SAC-spindle assembly checkpoint; CIN-chromosomal instability; NHEJ-non-homologous end joining; HR-homologous recombination; EMT-epithelial-mesenchymal transition.

It is tempting to speculate, that the ability of TRIP13 to inhibit apoptosis is of paramount importance in RCC patients. Firstly, it has been confirmed in renal tubular epithelial cells, the same that give rise to ccRCC. Secondly, there is accumulating evidence of positive responses to apoptosis inducers in RCC [33-40].

Disclosure of conflict of interest

None.